A major emphasis of this laboratory is to unravel the mechanism whereby the UNC-4 homeoprotein regulates synaptic specificity in the motor neuron circuit. In unc-4
) mutants, the usual inputs to VA motor neurons from AVA, AVD, and AVE command interneurons are replaced with gap junctions from AVB, which are normally reserved for VB motor neurons. The unc-4
wiring defect was initially detected by John White and colleagues by EM reconstruction of serial sections. We are now developing an alternative approach to detect motor neuron-specific synapses in living animals in the confocal microscope. The idea is to label presynaptic and postsynaptic proteins with different colored GFPs and then to use specific promoters to drive expression of these GFP-tagged markers in the command interneurons and in their postsynaptic motor neuron partners. The Zeiss LSM 510 confocal microscope provides argon laser lines that are ideal for discrete excitation of CFP (458 nm) and YFP (514 nm). An authentic synapse in the C. elegans motor neuron circuit labeled in this manner should appear as superimposed CFP and YFP "spots" in the ventral nerve cord. We have constructed YFP-tagged synaptobrevin (SNB-1), a membrane component of neurotransmitter vesicles, and used the nmr-1
promoter to drive expression in AVA and AVD command interneurons. Animals expressing the nmr-1
-SNB-1-YFP transgene show YFP puncta in the ventral nerve cord. We are now testing candidate proteins for marking the postsynaptic membranes in motor neurons. The vertebrate protein spinophilin (aka, neurabin II) interacts with actin microfilaments and is concentrated at the postsynaptic membrane where it is believed to tether Protein Phosphatase I (PP1) for modulation of glutamate receptor activity. Neurabin II is also more widely expressed in vertebrate tissues and may interact with additional classes of cytoplasmic proteins and receptors at the cell surface. A related vertebrate homolog, neurabin I, is largely expressed in the brain. C. elegans contains a single neurabin-like gene, nab-1
[C43E11.6, P = 5 x e-49]. We fused GFP to the NAB-1 C-terminus at a site that does not perturb localization of spinophilin in mammalian neurons. Transgenic animals expressing GFP-tagged NAB-1 under the control of nab-1
gene regulatory sequences show punctate staining in the nerve ring and in axial nerve cords, a pattern that is strikingly similar to that of other GFP-tagged synaptic proteins. We also see transient GFP-localization to hypodermal cells midway through embryogenesis. The goal now is to determine if NAB-1 localizes to the motor neuron postsynaptic membrane and then to confirm that a GFP-tagged version of NAB-1 can be used to score specific synapses between command interneurons and motor neurons in the ventral nerve cord.